A goal of regenerative medicine is to regenerate the architecture and function of tissues and organs totally or partially lost due to disease, trauma and ageing. Stem cells are considered crucial building blocks for any regenerative strategy. The challenge and motivation are to find ways for recruiting and/or delivering to the injured site pluripotent stem cells populations capable of regenerating nonfunctional or lost tissues and organs. Bone marrow and to a very limited extent peripheral blood, fat, and muscle are the major sources for such a population. A serious drawback of these sources is that aging and disease substantially lower the functionality and possibly the availability of adult stem cells.
Mesenchymal stem cells (MSCs) were suggested for regenerative therapy in the diseases involving neurodegeneration (Barzilay, R., Levy, Y. S., Isr Med Assoc J, 2006, 8, 61-66, Blondheim, N. R., et al., Stem Cells Dev., 2006, 15, 141-164; Sadan, O., Melamed, E. & Offen, D. Expert Opin Biol Ther., 2009, 9, 1487-1497).
Acute or chronic damage to the nervous system includes a variety of conditions such as motion disorders, dissociative disorders, mood disorders, affective disorders, addictive disorders and convulsive disorders. A chronic neurological disease or disorder is for example, Parkinson's disease (PD), multiple sclerosis, epilepsy, amyotrophic lateral sclerosis, glaucomatous neuropathy, Alzheimer's disease and Huntington's disease. Other demyelinating or dysmyelinating disorders, such as Pelizaeus-Merzbacher disease, leukodystrophies, neuritis and neuropathies are also categorized as CNS disorders. An acute neurological disease or disorder is for example stroke or autoimmune encephalomyelitis. A CNS disease or disorder may be a psychiatric disease or disorder such as schizophrenia, anxiety, depression or autism.
US 2006/211109 describes improved methods for efficiently producing neuroprogenitor cells and differentiated neural cells such as dopaminergic neurons and serotonergic neurons from pluripotent stem cells, for example human embryonic stem cells.
Parkinson's disease is a chronic progressive neurodegenerative disease that affects over 1% of the population over 65 years and has been positioned as the second most common neurodegenerative disorder after Alzheimer's Disease. The cardinal symptoms of PD include resting tremor, rigidity, bradykinesia, and postural instability. Advanced symptoms also include non-motor signs such as autonomic, sensory, psychiatric and cognitive impairments (Arenas, E., 2010 Biochem Biophys Res Commun 396, 152-156). The clinical motor dysfunction observed in PD is primarily the consequence of a progressive and selective degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta of the ventral midbrain, resulting in a severe deficiency of dopamine in the nigrostriatal pathway affecting the striatum. Many therapeutic approaches, including cell therapy, have been tested in PD therapy but none was found successful to date. (Lindvall, O. & Kokaia, Z., J Clin Invest., 2010 120, 29-402208).
PD is an attractive model disease to study the effect of direct cell replacement therapy (CRT) by new and healthy neurons (Ganz J, et al., Expert Rev Neurother. 2011; 11:1325-1339 Although this approach was exhaustively investigated, several obstacles have emerged that prevented its wide application in PD therapy.
The progress made in stem cell biology enabled the development of new approaches aimed at coaxing various stem cells to differentiate into DA neurons. These include genetic manipulation and exposure to a variety of morphogenetic factors or chemical compounds. A myriad of stem cell populations have been explored in the search for new DA neuron sources including embryonic, mesenchymal, neural progenitors, induced pluripotent stem cells as well as induced neuronal cells (Wernig M, et al. Proc Natl Acad Sci USA. 2008; 105:5856-5861; Caiazzo M, et al. Nature. 2011; 476:224-227).
Another stem cell based therapy approach currently under research is to use the cells as vectors that contain and secrete neuroprotective or neurotrophic agents, in order to provide neuroprotection or trophic stimuli to the surviving neurons. In the nervous system, the neuroprotective role is mostly played by astrocytes, also called astroglia, which are one of the major types of glial cells and historically have been regarded as the support cells of the nervous system (Kimelberg H K, Nedergaard M. 2010; 7:338-353). Recently malfunction of astrocytes has been proposed to play a role in the pathogenesis of non-cell autonomous diseases (Miguel E, et al. PLoS One. 2012; 7:e34776; Diaz-Amarilla P, et al. Proc Natl Acad Sci USA. 2011; 108:18126-18131).
Neurotrophic factors (NTFs) are naturally occurring polypeptides that support the development, survival and neurite outgrowth in neurons and have been related to neurotransmitter production and release (Gritti A, Bonfanti L. Neuron Glia Biol. 2007; 3:309-323). Several studies concerning neuronal injury have demonstrated that NTFs such as brain-derived neurotrophic factor (BDNF), Vascular endothelial growth factor (VEGF), glial-derived neurotrophic factor (GDNF) and insulin growth factor-I (IGF-1) play an important role in the development, maintenance and regeneration of the nervous system (Dadon-Nachum M, et al. Stem Cell Rev. 2011; 7:664-671; Xiong N, et al. Gene Ther. 2011; 18:394-402). NTFs administration in neuronal dysfunction animal models, for instance, in motor neuron diseases, has been related to axon regeneration and functional recovery (Zheng C, et al. Biochem Biophys Res Commun. 2007; 363:989-993). Although there are some indications of restoration and recovery of motor function, clinical trials of systemic or intrathecal administration of recombinant NTFs to patients with motor neuron disorders did not show significant efficacy, possibly due to NTFs short half-life, poor delivery and low concentrations at target sites. Thus, continuous cellular-derived supply of NTF may overcome this drawback and provide an efficient treatment modality. Transplantation of stem/progenitor cells that mature into astrocytes-like cells in vivo have been reported to improve the outcome of spinal cord injury and ALS symptoms in mice models (Jin Y, et al. J Neurotrauma. 2011; 28:579-594). Genetically modified cells, induced to overexpress NTFs, enhanced nerve regeneration and preserved neuromuscular junctions in motorneuron lesions, such as spinal cord injury and ALS (Dadon-Nachum M et al. Journal of Stem Cells and Regenerative Medicine. 2012; 8:22-26).
It was shown that after a two-step medium based protocol and without transgenes expression, MSCs can be induced into NTFs secreting cells with astrocyte-like characteristics. After transplantation of these cells, clinical symptoms are attenuated in mouse models of multiple sclerosis, Parkinson's disease, Huntington, optic nerve transection and sciatic nerve injury (Sadan O, et al. Exp Neurol. 2012; 234:417-427). MSC differentiated into astrocyte-like cells, show the ability to protect motorneurons from sciatic nerve injury by preserving rat neuromuscular junctions (NMJ) and by increasing the NTFs levels at the site of injury.
WO2004/046348 teaches differentiation protocols for the generation of neural-like cells from bone marrow-derived stem cells.
WO2006/134602 teaches differentiation protocols for the generation of neurotrophic factor secreting cells.
WO2007/066338 teaches differentiation protocols for the generation of oligodendrocyte-like cells.
WO2009/144718 discloses stem cells having mesenchymal phenotype and increased secretion of brain-derived neurotrophic factor (BDNF). Bone-marrow derived cells, are propagated in platelet lysate prior to differentiation to achieve increases secretion of neurotrophic factors.
WO2010/090843 discloses gingiva-derived mesenchymal stem cells and their use in immunomodulation and reconstruction.
WO 2012/009581 discloses pharmaceutical compositions comprising gingiva-derived mesenchymal stem cells and methods of treating inflammation, wound healing and contact hypersensitivity.
Oral Mucosa Stem Cells
Oral Mucosa is the mucosal lining the oral cavity, namely: the cheeks and the alveolar ridge including the gingiva and the palate, the tongue, the floor of the mouth and the oral part of the lips. Oral mucosa consists of an epithelial tissue of ectodermal origin and the lamina propria (LP) which is a connective tissue of ectomesenchymal origin. Similarly to the ectomesenchymal origin of connective tissues in the oral cavity, cells of the oral mucosa lamina propria (OMLP) originate from the embryonic ectodermal neural crest. Wounds in human oral mucosa heal mainly by regeneration. The rate of healing is faster than that in the skin or other connective tissues and seems to be affected negligibly by age and gender (Szpaderska, A. M., et al., J Dent Res, 2003, 82, 621-626). Recently, the first evidence that the OMLP gives rise to a robust multipotent SC population was provided suggested the human oral mucosa as a novel source for therapeutic adult SC (Marynka-Kalmani, K., et al., Stem Cells, 2010, 28, 984-995). They also reported that explantation of the adult human OMLP reproducibly generates trillions of SC that they called, human oral mucosa stem cells (hOMSC). Immunophenotyping of hOMSC revealed a primitive neural crest stem cells (NCSC) phenotype, which is not affected by adult donor age. The expression of early neural crest stem/progenitor cell markers (Sox2 and p75) in vitro and in vivo points to the neural crest origin of this population. The identification of mRNA for Oct4, Sox2, Myc-c, and Kfl4, the four transcription factors used for induced pluripotent stem cell (iPS) induction (Takahashi, K. & Yamanaka, S. Cell, 2006, 126, 663-676) in the human palate cells (Widera, D., et al. Stem Cells, 2009, 27, 1899-1910) suggests that OMLP harbors a primitive SC population.
In vitro assays demonstrated that unsorted hOMSC subjected to neuronal differentiation regimens, differentiated into neuroectoderm lineages as evidenced by the decrease in Oct4 and Nanog, increase in MAP2 expression (neural) and the induction of neuritogenesis in PC12 cells, the last being considered a functional assay for glial differentiation (Bampton E T, Taylor J S. J Neurobiol 2005; 63:29-48). Undifferentiated hOMSC however, supported only PC12 cells survival, probably via the secretion of nerve growth factor (NGF) and Fibroblast Growth Factor-2 (FGF-2). In addition, it was shown (Marynka-Kalmani, K., et al., ibid) that hOMSC can differentiate in vitro, into lineages of the three germ layers and after stimulation with dexamethasone, their implantation in vivo resulted in the formation of bilineage mixed tumors consisting of tissues that develop from cranial neural crest cells during embryogenesis. WO 2008/132722 discloses the lamina propria of the mucosa of the gastrointestinal tract and in particular of the oral mucosa, as a source for pluripotent adult stem cells.
There remains an unmet need to provide safe and readily accessible source of stem cells and methods for their differentiation into different types of neural cells. These stem cells should be capable of generating a population that can be expanded in vitro without losing its pluripotency and differentiated into which can be retransplanted into the affected donor to effectively restore neurological functions. Adult stem cells seems to take the lead and are being positioned as a safe alternative deprived from the immunologic, ethical and safety concerns associated with embryonic, fetal and induced pluripotent stem cell (iPS) stem cells.